In a mouse pituitary tumor cell line, AtT-20, secretion of ACTH and beta-endorphin is regulated by the presence or absence of a secretagogue. The ACTH and beta-endorphin are stored in dense secretory granules. The same cells have a second non-regulated pathway that secretes the polypeptide precursor pro-opiomelanocortin (POMC), and at least one membrane glycoprotein. We propose that only polypeptides with a particular sorting address are segregated into secretory granules and stored in the cell. Wtih the hope that a similar sorting address might be conserved and is present in all precursors of polypeptide hormones, we have introduced the genes for human insulin and human growth hormone into AtT-20 cells. Preliminary evidence suggests that the genes are expressed, stored in secretory granules and released on exposure to a secretagogue. Unlike other cells transfected with insulin DNA, however, the transfected AtT-20 cells release insulin, not proinsulin. These obervations have led us to propose the following experiments. Frist we plan to identify the sorting address by recombinant DNA and antibody techniques. Secondly, we plan to confirm that the processing enzymes in AtT-20 cells produce authentic insulin from proinsulin. Finally, we will make use of the nerve-like morphology of AtT-20 cells grown in 8-Br-cAMP to examine the question of how post-Golgi vesicles target themselves to their correct destination. These data should identify the sorting signals and molecules involved in segregating and packaging peptide hormones in endocrine tissue and how they might be deranged, especially in peptide-secreting tumors. At the same time we may learn how secreting cells make biologically active hormones out of prohormones.